Project Summary: The ability of cells to migrate in a directed manner is critical to a variety of biological processes including morphogenesis, would repair, immunological response and cancer progression. A key feature of migration in an in vivo setting is the ability to break through junctional barriers as a cell migrates across tissue boundaries. This ability requires a distinct set of molecular regulators and requires manipulation of cytoskeletal dynamics. We have developed the migration and radial intercalation of multi-ciliated cells (MCCs) and ionocytes (ICs) in the skin of Xenopus embryos as an experimentally pliable model system for addressing the molecular mechanisms involved as these cells break through the epithelium. Our data indicates that diverse regulators of microtubule (MT) stability have profound effects on the ability of these cells to break through junctional barriers. We have developed the molecular tools and imaging techniques to manipulate, visualize and quantifiably score the ability of MCCs and ICs to migrate towards the apical surface and intercalate into the outer epithelium. We will use these methods to address: (1) Establishment of stabilized MTs along the axis of migration, (2) Junctional remodeling during intercalation, (3) Small GTPase regulation of intercalation. The results from these experiments will provide a detailed molecular mechanism for the complex regulation of MT dynamics during migration and intercalation. While many of these experiments build on migration studies in cell culture, our preliminary data indicates that the in vivo 3 dimensional aspect of our proposed experiments will provide a novel paradigm for understanding this important biological problem. The ability to block cells from traversing junctional barriers could have a significant impact on human health as the ability of metastatic cancers cells to migrate through tissue barriers is a critical step in cancer progression. The unique ability to address the issue of intercalation in distinct cells types will allow us to identify core components of the process. Additionally, the profound reproducibility of intercalation during a discreet developmental window will allow us to identify subtle phenotypes in a quantifiably robust manner that would not be feasible in many other experimental systems.